Myosins are ubiquitous intracellular motors that use the energy of ATP hydrolysis to move on actin filaments. After myosin II was first characterized in vertebrate muscle, various other classes have been found. Class VI myosin has been shown recently to walk "backwards" on actin, i.e. in the direction opposite to that of other myosins. Subsequent studies revealed important transport and anchoring roles for this backward motor in a variety of cells. Several crystal structures of the motor in various conformations have been obtained recently. In this proposal, we intend to utilize these structures and other experimental data to elucidate the complete cycle and energetics of the myosin VI motor at the atomic level by means of numerical simulations. We will determine transition paths between the available crystal structures using Minimum Energy Path (MEP), Block Normal Mode (BNM) analysis, and steered/biased/targeted Molecular Dynamics. The potential of mean force associated with the transitions will be computed, which will give the free energy change associated with the transitions. We will determine the affinities of the crystal structures for the various ligands by free energy simulation methods to determine how different conformations are stabilized by the different ligands. To understand the differences between myosin VI and other (forward-stepping) myosins, we will compare the mechanisms of myosin VI and myosin V (research on the myosin V is currently being done in the Karplus laboratory). Many of the methods mentioned above have been implemented in the program CHARMM, which we will use for simulation and analysis. Combining our analysis of the motor head with available cryo-electron microscopy structures of the acto-myosin complex, single-molecule studies in the literature, and elasticity theory, we will develop a coarse-grained model for this unusual myosin. Myosin VI malfunction is associated with hereditary hearing loss, and myosin VI concentration is much higher in ovarian cancer cells than in normal cells. Results of our research could lead to the design of new therapies for these diseases. For example, an understanding of the mechanism of myosin VI can identify ways to regulate the activity of this motor.